pLV-hUbC-dCas9-KRAB-T2A-GFP plasmid used for CRISPR interference has been described previously (Esvald et al., 2020), and pLV-hUbC-VP64-dCas9-VP64-T2A-GFP plasmid used for CRISPR activation was obtained from Addgene (plasmid #59791). Lentiviral particles were produced as described previously (Koppel et al., 2018). Relative viral titers were estimated from provirus incorporation rate measured by qPCR, and equal amounts of functional viral particles were used for transduction in the following experiments. The efficiency of viral transduction was at least 90–95% based on EGFP expression in transduced cells.

Rat cortical neurons were transduced at 0 DIV, whereas cortical astrocytes were transduced after sub-culturing at 7 DIV. After treatments at 8 DIV for neurons or at 14 DIV for astrocytes, the cells were lysed and RNA was extracted with RNeasy Mini Kit (Qiagen, Hilden, Germany) using on-column DNA removal with RNase-Free DNase Set (Qiagen, Hilden, Germany). RNA concentration was measured with BioSpec-nano spectrophotometer (Shimadzu Biotech, Kyoto, Japan). cDNA was synthesized from equal amounts of RNA with Superscript III or Superscript IV reverse transcriptase (Invitrogen, Waltham, MA, USA) using oligo(dT)20 or a mixture of oligo(dT)20 and random hexamer primer (ratio 1:1, Microsynth, Balgach, Switzerland). To measure +3 kb enhancer eRNAs, cDNA was synthesized using a mixture of antisense eRNA-specific primer and Hprt1 primer (1:1 ratio). The primers used for cDNA synthesis are listed in Supplementary file 1.

All qPCR reactions were performed in 10 µl volume in triplicates with 1× HOT FIREpol EvaGreen qPCR Mix Plus (Solis Biodyne, Tartu, Estonia) and primers listed in Supplementary file 1 on LightCycler 480 PCR instrument II (Roche, Basel, Switzerland). Gene expression levels were normalized to Hprt1 mRNA levels in neurons and cyclophilin B mRNA levels in astrocytes. Data were log-transformed and autoscaled (as described in Vandesompele et al., 2002) for statistical analysis, mean and SEM were calculated, and data were back-transformed for graphical representation.

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